The trabecular meshwork is believed to be a major site for the regulation of the aqueous outflow. Residing in this tissue are trabecular meshwork cells that are essential for the maintenance of normal outflow. Aberrations of cell integrity may be a key step toward obstruction of the aqueous outflow, intraocular pressure elevations, and glaucomatous conditions. Besides the cells, extracellular matrix (ECM) materials such as fibronectin, laminin, and collagens are also thought to be important for normal functioning of the trabecular meshwork. We propose, in this application, to examine the human trabecular meshwork for the integrins, a family of receptors for ECM elements and other cell-surface proteins, that can mediate cell adhesion, influence cellular activities, and affect the growth, morphology, metabolism, and differentiation of cells. Specifically, the types and quantity of the receptors for fibronectin, laminin, and collagens in human trabecular meshwork tissues and in primary, first- and second-passage cell cultures will be determined by gel electrophoresis, affinity chromatography, immunoprecipitation, and other methods. Since primary open angle glaucoma (POAG) is known to be an age- related disorder, specimens obtained from donors of three different age groups (< 20, 21-49, and > 50 years) will be studied to determine whether variations exist with aging. Roles of integrins in attachment and spreading of cultured trabecular meshwork cells will also be evaluated. We will further assess the composition of collagens in human trabecular meshwork tissues and cell cultures. We have shown that collagen types I, III, IV, V, and VI are expressed in the human trabecular meshwork. Our investigations will be expanded to include studies for collagen type VIII and to systematically and quantitatively evaluate the age-related changes in the gene expression of collagens. Protocols for Northern blot, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), and biochemical analyses will be used. In addition, the effects of glucocorticoids on trabecular meshwork cells in both cell culture and organ culture systems will be evaluated. Glucocorticoids such as dexamethasone induced intraocular pressure elevations in susceptible humans and rabbits, and the underlying alterations in the trabecular meshwork have bene suggested to be similar to those seen in POAG. In our study, the modulation by dexamethasone in the production of fibronectin, laminin, collagens, and integrins will be assessed by techniques including immunostaining, ELISA, and Western blotting. Variations in mRNA levels will be documented by molecular biology methods. Moreover, changes induced by dexamethasone in levels of cathepsins B and G, elastase, and alpha1-proteinase inhibitor, all of which are pivotal for the turnover and remodeling of the ECM, will be investigated by immunohistochemical staining, biochemical assays, and ELISA. mRNA levels for cathepsin G and alpha1-proteinase inhibitor will also be measured to delineate the effects of dexamethasone. Our goal is to further elucidate the biologic characteristics of healthy as well as experimentally-altered trabecular meshwork cells, particularly with regard to the dynamics and control mechanisms involved in the synthesis and turnover of ECM proteins in the trabecular meshwork. This information will assist in uncovering the pathogenic processes of POAG.